Calcium channel; Permeability-glycoprotein (P-gp); CYP3A4[1]

Citric medicines limit the uptake of EverFluor FL Verapamil (EFV) by TR-iBRB2 cells, while verapamil inhibits the uptake in a concentration-dependent manner with an IC50 of 98.0 μM[4].

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Functional Analysis of EverFluor FL Verapamil (EFV) Uptake by TR-iBRB2 Cells [4]
The function of EFV uptake was investigated in TR-iBRB2 cells, and the linear increase in EFV uptake was observed for at least 10 min with an initial uptake rate of 65.3 ± 6.7 μL/(min·mg protein) (Fig. 4a). EFV uptake was significantly reduced by 57.8% at 4°C (Fig. 4a), and no significant change in EFV uptake was seen in the experiment with buffer in which Na+ was replaced by Li+ or K+ (Fig. 4b). The effect of the extracellular and intracellular pH on EFV uptake was investigated in TR-iBRB2 cells. The uptake of EFV at pH 6.4 and 8.4 exhibited no significant difference from that at pH 7.4 (Fig. 4c), whereas the acute treatment of TR-iBRB2 cells with NH4Cl produced a significant reduction in EFV uptake by 34% (Fig. 4d).

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Inhibition Analysis of EverFluor FL Verapamil (EFV) Uptake by TR-iBRB2 Cells [4]
The in vitro distribution analysis suggested the transport of EFV at the inner and outer BRB. In particular, the inner BRB has been known to nourish two-thirds of the retinal tissue. Therefore, the inhibitory effect of several compounds on EFV uptake by TR-iBRB2 cells was investigated (Table I), and cationic drugs, including desipramine, imipramine, propranolol and verapamil, markedly inhibited the uptake of EFV by more than 47%. Furthermore, cationic drugs, including quinidine, pyrilamine, and timolol, moderately inhibited EFV uptake by more than 25%, while no significant effect was produced by cimetidine, clonidine, amantadine, acetazolamide, choline, tetraethylammonium (TEA), 1-methyl-4-phenylpyridinium (MPP+), L-carnitine, serotonin and p-aminohippuric acid (PAH). In addition, the inhibition analysis of EFV uptake showed a concentration-dependent inhibition of EFV uptake by verapamil with an IC50 of 98.0 μM (Fig. 4e).

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Purpose: To investigate the blood-to-retina verapamil transport at the blood-retinal barrier (BRB).
Methods: EverFluor FL Verapamil (EFV) was adopted as the fluorescent probe of verapamil, and its transport across the BRB was investigated with common carotid artery infusion in rats. EFV transport at the inner and outer BRB was investigated with TR-iBRB2 cells and RPE-J cells, respectively.
Results: The signal of EverFluor FL Verapamil (EFV) was detected in the retinal tissue during the weak signal of cell impermeable compound. In TR-iBRB2 cells, the localization of EFV differed from that of LysoTracker® Red, a lysosomotropic agent, and was not altered by acute treatment with NH4Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH4Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH4Cl treatment during no significant effect obtained by different extracellular pH and V-ATPase inhibitor. The EFV uptake by TR-iBRB2 cells was inhibited by cationic drugs, and inhibited by verapamil in a concentration-dependent manner with an IC50 of 98.0 μM.
Conclusions: Our findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB [4].

Verapamil, when taken orally, can help control the atrioventricular nodal response in atrial fibrillation and prevent atrioventricular reentry tachycardia[2]. IV verapamil injections are given into femoral veins before ischemia occurs. The incidence of ventricular arrhythmias, such as ventricular tachycardia (VT), ventricular fibrillation (VF), and premature ventricular contractions (PVC), is significantly reduced by verapamil (1 mg/kg) for 45-minute coronary artery occlusion. When the heart experiences ischemia, the total arrhythmia score rises noticeably. The enhancement of total arrhythmia scores brought on by ischemia is significantly inhibited by verapamil (1 mg/kg)[5].

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The antiarrhythmic effects of verapamil were observed before it was appreciated that it was a calcium ion-antagonist. Intravenous verapamil is highly effective in the termination of paroxysmal reciprocating atrioventricular tachycardia, whether associated with preexcitation or involving the atrioventricular node alone. It consistently slows and regularises the ventricular response in atrial fibrillation, and usually increases the degree of AV-nodal block in atrial flutter though it occasionally induces a return to sinus rhythm. Given orally it is useful for the prophylaxis of atrioventricular reentry tachycardia, and also in modulating the atrioventricular nodal response in atrial fibrillation. Favourable response in ventricular tachycardia is exceptional and then seen in specific benign varieties. Verapamil is the agent of choice for the termination of paroxysmal supraventricular tachycardia.[2]

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Intervention: The patients were treated with either metoprolol (Seloken ZOC 200 mg o.d.) or verapamil (Isoptin Retard 240 b.i.d.). Acetylsalicylic acid, ACE inhibitors, lipid lowering drugs and long acting nitrates were allowed in the study. End points: Death, non-fatal cardiovascular events including acute myocardial infarction, incapacitating or unstable angina, cerebrovascular or peripheral vascular events. Psychological variables reflecting quality of life i.e. psychosomatic symptoms, sleep disturbances and an evaluation of overall life satisfaction. Results: Combined cardiovascular events did not differ and occurred in 30.8% and 29.3% of metoprolol and verapamil treated patients respectively. Total mortality in metoprolol and verapamil treated patients was 5.4 and 6.2%, respectively. Cardiovascular mortality was 4.7% in both groups. Non-fatal cardiovascular events occurred in 26.1 and 24.3% of metoprolol and verapamil-treated patients, respectively. Psychosomatic symptoms and sleep disturbances were significantly improved in both treatment groups. The magnitudes of change were small and did not differ between treatments. Life satisfaction did not change on either drug. Withdrawals due to side effects occurred in 11.1 and 14.6% respectively. Conclusion: This long term study indicates that both drugs are well tolerated and that no difference was shown on the effect on mortality, cardiovascular end points and measures of quality of life.[3]

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The present study was to test the hypothesis that anti-arrhythmic properties of verapamil may be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of verapamil. Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein [5].

Methods: EverFluor FL Verapamil (EFV) was adopted as the fluorescent probe of verapamil, and its transport across the BRB was investigated with common carotid artery infusion in rats. EFV transport at the inner and outer BRB was investigated with TR-iBRB2 cells and RPE-J cells, respectively. Results: The signal of EFV was detected in the retinal tissue during the weak signal of cell impermeable compound. In TR-iBRB2 cells, the localization of EFV differed from that of LysoTracker® Red, a lysosomotropic agent, and was not altered by acute treatment with NH4Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH4Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH4Cl treatment during no significant effect obtained by different extracellular pH and V-ATPase inhibitor. The EFV uptake by TR-iBRB2 cells was inhibited by cationic drugs, and inhibited by verapamil in a concentration-dependent manner with an IC50 of 98.0 μM[4].

The antiarrhythmic effects of verapamil were observed before it was appreciated that it was a calcium ion-antagonist. Intravenous verapamil is highly effective in the termination of paroxysmal reciprocating atrioventricular tachycardia, whether associated with preexcitation or involving the atrioventricular node alone. It consistently slows and regularises the ventricular response in atrial fibrillation, and usually increases the degree of AV-nodal block in atrial flutter though it occasionally induces a return to sinus rhythm. Given orally it is useful for the prophylaxis of atrioventricular reentry tachycardia, and also in modulating the atrioventricular nodal response in atrial fibrillation. Favourable response in ventricular tachycardia is exceptional and then seen in specific benign varieties. Verapamil is the agent of choice for the termination of paroxysmal supraventricular tachycardia[2].

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Confocal Microscopy of TR-iBRB2 Cells and RPE-J Cells [4]
TR-iBRB2 cells and RPE-J cells were immortalized rat retinal capillary endothelial cells and retinal pigment epithelial cells to be used as the inner and outer BRB model cell lines, and were seeded at 5 × 103 and 7 × 103 cells/well on BioCoat™ Collagen I Cellware 8-well culture slide, respectively, and cultured at 33°C under 5% CO2/air. After a 48 h cultivation, the uptake assay was initiated by adding extracellular fluid (ECF)-buffer containing EverFluor FL Verapamil (EFV) (1 μM) or LTR (300 nM for TR-iBRB2 cells, 600 nM for RPE-J cells), and these concentrations were set by referring previous reports. The assay was terminated at a designated time by washing cells with ice-cold ECF buffer three times. After fixation of the cells with 4% paraformaldehyde, the cells were subjected to confocal microscope observation with LSM700 as reported elsewhere.

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Cell Uptake Study [4]
In vitro uptake analysis using TR-iBRB2 cells was conducted by referring to previous reports, and cells were cultured on collagen-coated 24-well plates at 33°C under 5% CO2/air. Uptake assay was started by adding ECF-buffer containing EverFluor FL Verapamil (EFV) (1 μM in 200 μL) at 37°C, and was terminated by washing the wells three times with ice-cold ECF-buffer. After adding ECF-buffer (200 μL/well), cells were homogenized in an ultrasonic homogenizer, and the cell protein contents and the fluorescence intensity of EFV was measured with a multi-mode microplate reader system. EFV uptake was expressed as the cell-to-medium (C/M) ratio by means of Eq. 1. EverFluor FL Verapamil (EFV) uptake in the presence of inhibitors was expressed as the fluorescence intensity ratio (FI ratio) by means of Eq. 2. The nonlinear least-square regression analysis program, MULTI, was used for the determination of the 50% inhibitory concentration (IC50) for verapamil in EverFluor FL Verapamil (EFV) uptake, and the data were fitted to Eq. 3. In the in vitro inhibition analysis, the concentration of inhibitors was set to be 500 μM by referring our previous report on verapamil transport by TR-iBRB2 cells, P and Pmax are the FI ratios with and without inhibitors, and Pmin is the inhibitor-insensitive FI ratio with inhibitor. [I] and n are the inhibitor concentration and the Hill coefficient, respectively.

The present study was to test the hypothesis that anti-arrhythmic properties of Verapamil may be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of Verapamil . Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of Verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after Verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein[5].

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Common Carotid Artery Infusion Analysis [4]
In vivo distribution analysis of EverFluor FL Verapamil (EFV) to the retina was conducted by modifying the in situ brain perfusion method reported previously. In the anesthetized Wistar rats with pentobarbital (50 mg/kg), the right external carotid artery was ligated by a silk thread. After ligating the right common carotid artery, polyethylene tube was inserted into the right common carotid artery just below the bifurcation of the external carotid artery, and fixed by silk thread. The dosage conditions for fluorescent compounds were verified by referring previous reports that examined conditions without toxicity. Ringer-HEPES solution containing EverFluor FL Verapamil (EFV) (400 μg/3.5 mL), Rho-D (4 mg/3.5 mL) or LTR (400 μg/3.5 mL) warmed at 37°C was infused into the pterygopalatine artery and the internal carotid artery at a constant flow rate (0.85 mL/min) with an infusion pump, and this flow rate was set to avoid damaging the barrier structure with consideration for the blood-flow rate of the retina (0.7 mL/(min·g retina)). At the end of the infusion, the rats were decapitated, and their right eyeballs were immediately collected to be soaked in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 3 h, followed by soaking in PBS containing 30% sucrose at 4°C. The tissues were then fixed in the optimal cutting temperature compound, and tissue slices were prepared by means of a cryostat. Tissue slices mounted on glass slides were treated with 4′,6-diamidino-2-phenylindole (DAPI) and VECTASHIELD mounting medium, to be examined with a confocal microscope as described elsewhere. The excitation wavelength of 488 nm was used for EverFluor FL Verapamil (EFV), and 543 nm was used for LTR and Rho-D.

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In vivo Arrhythmia Study [5]
Verapamil (1 mg/kg) was injected i.v. into a femoral vein 10 min prior to ischemia. A sham group underwent the same surgical procedures, except the suture underneath the LAD was left untied. In another series of experiment, arrhythmia was induced by Bay K8644, an L-type calcium channel agonist, at a dose of 0.1 mg/kg given i.v. into the FV. Verapamil (1 mg/kg) was administered 10 min prior to Bay K8644. All injections were performed within 30 sec.

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Heart Isolation and Perfusion [5]
Each heart underwent an initial 10 min of baseline normal perfusion and was subjected to perfusion at 37°C for 45 min. The hearts were then randomly divided into three groups: Control group (normal calcium perfusion) (1.5 mmol/L), high calcium group (high calcium perfusion) (3.3 mmol/L) and Verapamil group (high calcium plus verapamil perfusion) (3.3 mmol/L calcium +3 µmol/L Verapamil ). For measurement of arrhythmias, the ECG was continuously monitored during the entire perfusion period and the incidence of arrhythmias was evaluated.

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Measurement of ECG and Determination of Arrhythmia Score [5]
Anti-arrhythmic properties of Verapamil were determined in an animal model of ischemia-induced arrhythmia or in the presence of Bay K8644 or heptanol or Gap 26, respectively. The occurrence of cardiac arrhythmias throughout the 45 min was compared by ECG recording. For analysis of arrhythmia in Langendorff-perfused rat heart, each rat heart was continuously monitored with a positive electrode attached to the heart and a negative electrode to the aorta. After 10 min of a baseline normal perfusion period, incidences of arrhythmias under different concentrations of Ca2+ in the initial 45 min of perfusion period were compared. To enable a good quantitative comparison, 45 min of an ischemia period were divided into 15 3-min intervals in an in vivo arrhythmia evaluation and 45 min of a perfusion period was divided into 15 3-min intervals in an ex vivo arrhythmia investigation. Arrhythmia scores were evaluated as described previously. PVC ≤10/3-min period was recorded as 0; 10–50 PVC/3-min period was recorded as 1; ≥50 PVC/3-min period was recorded as 2; 1 episode of VF/3-min period was recorded as 3; 2–5 episodes of VF/3-min period was recorded as 4; and ≥5 episodes of VF/3-min period was recorded as 5.

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Adult male Sprague-Dawley (SD) rats (250 350 g) are used. Verapamil (1 mg/kg) is injected i.v. into a femoral vein 10 min prior to ischemia. A sham group undergoes the same surgical procedures, except the suture underneath the LAD is left untied. In another series of experiment, arrhythmia is induced by Bay K8644, an L-type calcium channel agonist, at a dose of 0.1 mg/kg given i.v. into the FV. Verapamil (1 mg/kg) is administered 10 min prior to Bay K8644. All injections are performed within 30 sec

Rats

Absorption, Distribution and Excretion
More than 90% of orally administered verapamil is absorbed - despite this, bioavailability ranges only from 20% to 30% due to rapid biotransformation following first-pass metabolism in the portal circulation. Absorption kinetic parameters are largely dependent on the specific formulation of verapamil involved. Immediate-release verapamil reaches peak plasma concentrations (i.e. Tmax) between 1-2 hours following administration, whereas sustained-release formulations tend to have a Tmax between 6 - 11 hours. AUC and Cmax values are similarly dependent upon formulation. Chronic administration of immediate-release verapamil every 6 hours resulted in plasma concentrations between 125 and 400 ng/mL. Steady-state AUC0-24h and Cmax values for a sustained-release formulation were 1037 ng∙h/ml and 77.8 ng/mL for the R-isomer and 195 ng∙h/ml and 16.8 ng/mL for the S-isomer, respectively. Interestingly, the absorption kinetics of verapamil are highly stereospecific - following oral administration of immediate-release verapamil every 8 hours, the relative systemic availability of the S-enantiomer compared to the R-enantiomer was 13% after a single dose and 18% at steady-state.
Approximately 70% of an administered dose is excreted as metabolites in the urine and ≥16% in the feces within 5 days. Approximately 3% - 4% is excreted in the urine as unchanged drug.
Verapamil has a steady-state volume of distribution of approximately 300L for its R-enantiomer and 500L for its S-enantiomer.
Systemic clearance following 3 weeks of continuous treatment was approximately 340 mL/min for R-verapamil and 664 mL/min for S-verapamil. Of note, apparent oral clearance appears to vary significantly between single dose and multiple-dose conditions. The apparent oral clearance following single doses of verapamil was approximately 1007 mL/min for R-verapamil and 5481 mL/min for S-verapamil, whereas 3 weeks of continuous treatment resulted in apparent oral clearance values of approximately 651 mL/min for R-verapamil and 2855 mL/min for S-verapamil.
/MILK/ Breast milk: Verapamil may appear in breast milk.
/MILK/ Verapamil is excreted into breast milk. A daily dose of 240 mg produced milk levels that were approx 23% of maternal serum. Serum levels in the infant were 2.1 ng/mL but could not be detected (<1 ng/mL) 38 hr after treatment was stopped. ... In a second case, a mother was treated with 80 mg 3 times/day for hypertension for 4 wk prior to the determination of serum & milk concns. Steady-state concentrations of verapamil and the metabolite, norverapamil, in milk were 25.8 and 8.8 ng/mL, respectively. These values were 60% and 16% of the concns in plasma. The investigators estimated that the breast-fed child received <0.01% of the mother's dose. Neither verapamil nor the metabolite could be detected in the plasma of the child.
The pharmacokinetics and hemodynamic effects of a combination of verapamil and trandolapril were studied in 20 patients with hypertension (ages 29-71 yr), 10 of whom also had fatty liver disease, who received a sustained-release oral capsule containing 180 mg verapamil and 1 mg trandolapril once daily for 7 days. For verapamil, no statistically significant differences were seen between patients with and without fatty liver with regard to Cmax (110.5 vs 76.5 ug/L), plasma AUC from 0-24 hr (1260.6 vs 941.2 ug/L hr), and elimination half-life (9.8 vs 9.2 hr).
An open, randomized, single dose study of the effects of food on the bioavailability of sustained-release (SR) verapamil hydrochloride (Isoptin) was conducted in 12 healthy volunteers (aged 19-65 yr) who received 240 mg of the SR preparation while fasting or with food and a conventional preparation while fasting. Although the elimination half-life of SR verapamil was unchanged, the time to maximum concentration was prolonged and the area under the concentration-time curve (AUC) was 80% of the regular preparation. Concomitant food administration prolonged the time to maximum concentration from 7.3+-3.4 to 11.7+-6.3 h but had little effect on the maximum concentration, half-life or AUC of SR verapamil.
For more Absorption, Distribution and Excretion (Complete) data for Verapamil (21 total), please visit the HSDB record page.

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Metabolism / Metabolites
Verapamil is extensively metabolized by the liver, with up to 80% of an administered dose subject to elimination via pre-systemic metabolism - interestingly, this first-pass metabolism appears to clear the S-enantiomer of verapamil much faster than the R-enantiomer. The remaining parent drug undergoes O-demethylation, N-dealkylation, and N-demethylation to a number of different metabolites via the cytochrome P450 enzyme system. Norverapamil, one of the major circulating metabolites, is the result of verapamil's N-demethylation via CYP2C8, CYP3A4, and CYP3A5, and carries approximately 20% of the cardiovascular activity of its parent drug. The other major pathway involved in verapamil metabolism is N-dealkylation via CYP2C8, CYP3A4, and CYP1A2 to the D-617 metabolite. Both norverapamil and D-617 are further metabolized by other CYP isoenzymes to various secondary metabolites. CYP2D6 and CYP2E1 have also been implicated in the metabolic pathway of verapamil, albeit to a minor extent. Minor pathways of verapamil metabolism involve its O-demethylation to D-703 via CYP2C8, CYP2C9, and CYP2C18, and to D-702 via CYP2C9 and CYP2C18. Several steps in verapamil's metabolic pathway show stereoselective preference for the S-enantiomer of the given substrate, including the generation of the D-620 metabolite by CYP3A4/5 and the D-617 metabolite by CYP2C8.
Metabolites: The main metabolite is norverapamil which has an elimination half-life very similar to that of the parent compound, ranging from 4 to 8 hours. Verapamil undergoes an extensive hepatic metabolism. Due to a large hepatic first-pass effect, bioavailability does not exceed 20 - 35% in normal subjects. Twelve metabolites have been described. The main metabolite is norverapamil and the others are various N- and 0-dealkylated metabolites. Elimination by route of exposure: Kidney: About 70% of the administered dose is excreted in urine within 5 days as metabolites, of which 3-4% is excreted as unchanged drug. Feces: About 16% of the ingested dose is excreted within 5 days in feces as metabolites. Breast milk: Verapamil may appear in breast milk.
Verapamil yields in the dog: 5-(3,4-dimethoxyphenethylamino)-2 -(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile; 2-(3,4-dimethoxyphenyl)-5 -(n-(4-hydroxy-3-methoxyphenethyl)methylamino)-2-isopropylvaleronitrile, and 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-methylaminovaleronitrile. The latter was also found in rats. /From table/ /salt not specified/
Verapamil and its major metabolite norverapamil were identified to be both mechanism-based inhibitors and substrates of CYP3A and reported to have non-linear pharmacokinetics in clinic. Metabolic clearances of verapamil and norverapmil as well as their effects on CYP3A activity were firstly measured in pooled human liver microsomes. The results showed that S-isomers were more preferential to be metabolized than R-isomers for both verapamil and norverapamil, and their inhibitory effects on CYP3A activity were also stereoselective with S-isomers more potent than R-isomers. A semi-physiologically based pharmacokinetic model (semi-PBPK) characterizing mechanism-based auto-inhibition was developed to predict the stereoselective pharmacokinetic profiles of verapamil and norverapamil following single or multiple oral doses. Good simulation was obtained, which indicated that the developed semi-PBPK model can simultaneously predict pharmacokinetic profiles of S-verapamil, R-verapamil, S-norverapamil and R-norverapamil. Contributions of auto-inhibition to verapamil and norverapamil accumulation were also investigated following the 38th oral dose of verapamil sustained-release tablet (240 mg once daily). The predicted accumulation ratio was about 1.3-1.5 fold, which was close to the observed data of 1.4-2.1-fold. Finally, the developed semi-PBPK model was further applied to predict drug-drug interactions (DDI) between verapamil and other three CYP3A substrates including midazolam, simvastatin, and cyclosporine A. Successful prediction was also obtained, which indicated that the developed semi-PBPK model incorporating auto-inhibition also showed great advantage on DDI prediction with CYP3A substrates.
The biotransformation pathway of verapamil, a widely prescribed calcium channel blocker, was investigated by electrochemistry (EC) coupled online to liquid chromatography (LC) and electrospray mass spectrometry (ESI-MS). Mimicry of the oxidative phase I metabolism was achieved in a simple amperometric thin-layer cell equipped with a boron-doped diamond (BDD) working electrode. Structures of the electrochemically generated metabolites were elucidated on the basis of accurate mass data and additional MS/MS experiments. We were able to demonstrate that all of the most important metabolic products of the calcium antagonist including norverapamil (formed by N-demethylation) can easily be simulated using this purely instrumental technique. Furthermore, newly reported metabolic reaction products like carbinolamines or imine methides become accessible. The results obtained by EC were compared with conventional in vitro studies by conducting incubations with rat as well as human liver microsomes (RLMs, HLMs). Both methods showed good agreement with the data from EC/LC/MS. Thus, it can be noted that EC is very well-suited for the simulation of the oxidative metabolism of verapamil. In summary, this study confirms that EC/LC/MS can be a powerful tool in drug discovery and development when applied complementary to established in vitro or in vivo approaches.
Mechanism-based inactivation (MBI) of cytochrome P450 (CYP) 3A by verapamil and the resulting drug-drug interactions have been studied in vitro, but the inhibition of verapamil on its own metabolic clearance in clinic, namely auto-inhibition of verapamil metabolism, has never been reproduced in vitro. This paper aimed to evaluate the utility of gel entrapped rat hepatocytes in reflecting such metabolic auto-inhibition using hepatocyte monolayer as a control. Despite being a similar concentration- and time-dependent profile, auto-inhibition of verapamil metabolism showed apparent distinctions between the two culture models. Firstly, gel entrapped hepatocytes were more sensitive to such inhibition, which could be largely due to their higher CYP3A activity detected by the formation rates of 6-beta-hydroxy testosterone and 1'-hydroxy midazolam. Furthermore, the inhibitory effect of ketoconazole and verapamil on CYP 3A activity as well as the reduction of verapamil intrinsic clearance (CL(int)) by ketoconazole was only observed in gel-entrapped hepatocytes. In this respect, the involvement of CYP3A in auto-inhibition of verapamil metabolism could be illustrated in gel-entrapped hepatocytes but not in hepatocyte monolayer. All of these results indicated that hepatocytes of gel entrapment reflected more of verapamil metabolic auto-inhibition than hepatocyte monolayer and could serve as a suitable system for investigating drug metabolism.
Verapamil has known human metabolites that include 2-(3,4-dimethoxyphenyl)acetaldehyde, Norverapamil, D-702, M9 (D-703), and D-617.
Route of Elimination: Approximately 70% of an administered dose is excreted as metabolites in the urine and 16% or more in the feces within 5 days. About 3% to 4% is excreted in the urine as unchanged drug.
Half Life: 2.8-7.4 hours

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Biological Half-Life
Single-dose studies of immediate-release verapamil have demonstrated an elimination half-life of 2.8 to 7.4 hours, which increases to 4.5 to 12.0 hours following repetitive dosing. The elimination half-life is also prolonged in patients with hepatic insufficiency (14 to 16 hours) and in the elderly (approximately 20 hours). Intravenously administered verapamil has rapid distribution phase half-life of approximately 4 minutes, followed by a terminal elimination phase half-life of 2 to 5 hours.
The pharmacokinetics of verapamil and its metabolite, norverapamil, were studied in 10 patients (ages 19-69 yr) with portal hypertension and in 6 healthy subjects (ages 21-69 yr) who received an oral dose of 80 mg verapamil hydrochloride (Isoptin). The terminal phase half-life of verapamil was 210 hr in controls and 1384 hr in patients.
A toxicokinetic study performed in two cases showed plasma half lives of 7.9 and 13.2 hours, total body clearances of 425 and 298 mL/min. ...

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In Vivo Distribution Analysis of EverFluor FL Verapamil (EFV) to the Retina [4]
Common carotid artery infusion was conducted to investigate the distribution of EFV to the retina. In confocal microscopy, the signal of EFV (green) was uniformly detected in the region from inner limiting membrane (ILM) to the outer plexiform layer (OPL) (Fig. 1a), while a weak fluorescence signal was detected for Rho-D which is a cell impermeable substrate (data not shown). In addition, a strong fluorescence signal of EFV was detected in the photoreceptor outer segment (POS) (Fig. 1a), and the punctate signal of EFV was partially detected in the RPE (Fig. 1b, arrow head). In Vitro Distribution Analysis of EverFluor FL Verapamil (EFV) [4]
Confocal microscopy was performed to investigate the subcellular localization of EFV in the model cell lines of the inner and outer BRB. In TR-iBRB2 cells, an in vitro model cell line of the inner BRB, the fluorescence signal of EFV was detected all over the cells, while the fluorescence signal of LTR had a punctate distribution pattern, showing that subcellular distribution pattern of EFV is different from that of LTR (Fig. 2a). In the case of acute treatment with NH4Cl, no prominent change was seen in the subcellular localization of EFV, while the punctate distribution pattern of LTR was reduced (Fig. 2b). In addition, EFV uptake by TR-iBRB2 cells was not significantly changed in the presence of bafilomycin A1, an inhibitor of vacuolar-type H+-ATPase (V-ATPase) (Fig. 2c). [4]
In RPE-J cells, a punctate distribution pattern of LTR was observed, and this was reduced by acute treatment with NH4Cl (Fig. 3). The signal of EFV was observed all over the cells with a partial punctate distribution pattern, that merged with the punctate distribution pattern of LTR (Fig. 3a, arrow head), and this partial distribution was reduced by acute treatment with NH4Cl (Fig. 3).

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Limited information indicates that maternal doses of verapamil up to 360 mg daily produce low levels in milk. Newborns may have detectable verapamil serum levels, but levels are low. Verapamil would not be expected to cause any adverse effects in breastfed infants, especially if the infant is older than 2 months.
◉ Effects in Breastfed Infants
No adverse reactions have been reported among 3 infants aged 13 days, 8 weeks and 3 months who were exposed to verapamil in breastmilk.
◉ Effects on Lactation and Breastmilk
Verapamil can cause hyperprolactinemia and galactorrhea. The clinical relevance of these findings in nursing mothers is not known. The maternal prolactin level in a mother with established lactation may not affect her ability to breastfeed.

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Toxicity Summary
IDENTIFICATION AND USE: Verapamil is the drug of choice for prevention and treatment of paroxysmal supraventricular tachycardia. Verapamil has been shown to be effective in the treatment of angina pectoris. Verapamil may be used as an alternative treatment for mild or moderate hypertension. HUMAN STUDIES: Verapamil has a vasodilating action on the vascular system. Toxic effects occur usually after a delay of 1 to 5 hours following ingestion. After IV injection, symptoms appear after a few minutes. The main cardiovascular symptoms are: bradycardia and atrioventricular block (in 82% of cases) hypotension and cardiogenic shock (in 78% of cases) cardiac arrest (in 18% of cases). Pulmonary edema may occur. Impairment of consciousness and seizures may occur and are related to a low cardiac output. Nausea and vomiting may be observed. Metabolic acidosis due to shock and hyperglycemia may occur. Verapamil is a calcium channel blocker and inhibits the entry of calcium through calcium channels into cardiovascular cells. Verapamil reduces the magnitude of the calcium current entry and decreases the rate of recovery of the channel. Verapamil decreases peripheral vascular and coronary resistance but it is a less potent vasodilator than nifedipine. In contrast, its cardiac effects are more prominent than those of nifedipine. At doses necessary to produce arterial vasodilatation, verapamil has much greater negative chronotropic, dromotropic and inotropic effects than nifedipine. At toxic doses, calcium channel inhibition by verapamil results in three principal effects: hypotension due to arterial vasodilatation, cardiogenic shock secondary to a negative inotropic effect, bradycardia and atrio-ventricular block. The therapeutic effects of verapamil on hypertension and angina pectoris are due to arterial systemic and coronary vasodilatation. The antiarrhythmic activity of verapamil is due to a delay in impulse transmission through the AV node by a direct action. Toxicity may occur after ingestion of 1 g. Verapamil was tested on human peripheral lymphocytes in vitro using micronucleus (MN) test. The MN frequencies showed increase after all treatment. The results of FISH analysis suggest that verapamil, separately or combined with ritodrine, shows to a larger extent aneugenic than clastogenic effect. ANIMAL STUDIES: Verapamil promotes atrial fibrillation in normal dogs. In swine, verapamil toxicity, as defined by a mean arterial pressure of 45% of baseline, was produced following an average verapamil infusion dose of 0.6 +/- 0.12 mg/kg. This dose produced an average plasma verapamil concentration of 728.1 +/- 155.4 ug/L. Hypertonic sodium bicarbonate reversed the hypotension and cardiac output depression of severe verapamil toxicity in a swine model. ECOTOXICITY STUDIES: Effects of long-term exposure of verapamil on mutagenic, hematological parameters and activities of the oxidative enzymes of Nile tilapia, Oreochromis niloticus were investigated for 60 days exposure at the concentrations of 0.29, 0.58 and 1.15 mg/L in the fish liver. The exposure resulted in significantly high micronuclei induction of peripheral blood cells. The indices of oxidative stress biomarkers (lipid peroxidation and carbonyl protein) showed elevated level. There was increase in the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST). In other experiments, exposure to sub-lethal concentrations of verapamil (0.14, 0.29 and 0.57 mg/L) for period of 15, 30, 45 and 60 days, led to inhibition of acetylcholinesterase activities in the brain and muscle of the fish. Transcription of catalase (CAT), superoxide dismutase (SOD) and heat shock proteins 70 (hsp70) were up-regulated in both the tissues after the study period. In Carassius auratus, the behavioral alterations were observed in the form of respiratory difficulty and loss of body balance confirming the cardiovascular toxicity caused by verapamil at higher doses. In addition to affecting the cardiovascular system, verapamil also showed effects on the nervous system in the form of altered expression of parvalbumin. Acute exposure to verapamil significantly reduced the heart rate in the embryos and larvae of common carp (Cyprinus carpio). In the D. magna chronic toxicity test, several parameters, such as the survival percentage, the body length of D. magna, the time of first reproduction, and the number of offspring per female, were adversely affected during the exposure to 4.2 mg/L verapamil. During the 24-hr short-term exposure, verapamil caused a downregulated expression of the CYP4 and CYP314 genes. During the 21-day long-term exposure, verapamil significantly reduced the expression level of the Vtg gene, a biomarker of the reproduction ability in an oviparous animal.
Verapamil inhibits voltage-dependent calcium channels. Specifically, its effect on L-type calcium channels in the heart causes a reduction in ionotropy and chronotropy, thuis reducing heart rate and blood pressure. Verapamil's mechanism of effect in cluster headache is thought to be linked to its calcium-channel blocker effect, but which channel subtypes are involved is presently not known.

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Toxicity Data
LD50: 8 mg/kg (Intravenous, Mouse) (A308)

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Interactions
Drug interactions: protein-bound drugs
Drug Interactions: beta-adrenergic blocking agents
Drug Interactions: digoxin
Drug Interactions: hypotensive agents
For more Interactions (Complete) data for Verapamil (42 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Mouse ip 68 mg/kg /Verapamil hydrochloride/
LD50 Rat ip 67 mg/kg /Verapamil hydrochloride/
LD50 Rat oral 114 mg/kg /Verapamil hydrochloride/
LD50 Mouse iv 7.6 mg/kg /Verapamil hydrochloride/
For more Non-Human Toxicity Values (Complete) data for Verapamil (14 total), please visit the HSDB record page.

[1]. Verapamil as a culprit of palbociclib toxicity. J Oncol Pharm Pract. 2019 Apr;25(3):743-746.

[2]. Krikler DM. Verapamil in arrhythmia. Br J Clin Pharmacol. 1986;21 Suppl 2:183S-189S.

[3]. Effects of metoprolol vs verapamil in patients with stable angina pectoris. The Angina Prognosis Study in Stockholm (APSIS). Eur Heart J. 1996 Jan;17(1):76-81.

[4]. Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier. Pharm Res. 2018 Mar 12;35(5):93.

[5]. Anti-arrhythmic effect of Verapamil is accompanied by preservation of cx43 protein in rat heart. PLoS One. 2013 Aug 12;8(8):e71567.

Verapamil Hydrochloride is the hydrochloride form of Verapamil, which is a phenylalkylamine calcium channel blocking agent. Verapamil inhibits the transmembrane influx of extracellular calcium ions into myocardial and vascular smooth muscle cells, causing dilatation of the main coronary and systemic arteries and decreasing myocardial contractility. This agent also inhibits the drug efflux pump P-glycoprotein which is overexpressed in some multi-drug resistant tumors and may improve the efficacy of some antineoplastic agents. (NCI04)
A calcium channel blocker that is a class IV anti-arrhythmia agent.
See also: Verapamil (has active moiety); Trandolapril; verapamil hydrochloride (component of).

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Therapeutic Uses
Anti-Arrhythmia Agents; Calcium Channel Blockers; Vasodilator Agents
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each ClinicalTrials.gov record presents summary information about a study protocol and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where the study is being conducted; Contact information for the study locations; and Links to relevant information on other health Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly articles in the field of medicine. Verapamil hydrochloride is included in the database.
Oral calcium-channel blocking agents are considered the drugs of choice for the management of Prinzmetal variant angina. A nondihydropyridine calcium-channel blocker (e.g., diltiazem, verapamil) also has been recommended in patients with unstable angina who have continuing or ongoing ischemia when therapy with beta-blocking agents and nitrates is inadequate, not tolerated, or contraindicated and when severe left ventricular dysfunction, pulmonary edema, or other contraindications are not present. In the management of unstable or chronic stable angina pectoris, verapamil appears to be as effective as beta-adrenergic blocking agents (e.g., propranolol) and/or oral nitrates. In unstable or chronic stable angina pectoris, verapamil may reduce the frequency of attacks, allow a decrease in sublingual nitroglycerin dosage, and increase the patient's exercise tolerance. /Included in US product label/
Verapamil is used for rapid conversion to sinus rhythm of paroxysmal supraventricular tachycardia (PSVT), including tachycardia associated with Wolff-Parkinson-White or Lown-Ganong-Levine syndrome; the drug also is used for control of rapid ventricular rate in nonpreexcited atrial flutter or fibrillation. The American College of Cardiology/American Heart Association/Heart Rhythm Society (ACC/AHA/HRS) guideline for the management of adult patients with supraventricular tachycardia recommends the use of verapamil in the treatment of various SVTs (e.g., atrial flutter, junctional tachycardia, focal atrial tachycardia, atrioventricular nodal reentrant tachycardia (AVNRT)); in general, IV verapamil is recommended for acute treatment, while oral verapamil is recommended for ongoing management of these arrhythmias. /Included in the US product label/
For more Therapeutic Uses (Complete) data for Verapamil (14 total), please visit the HSDB record page.

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Drug Warnings
...Concurrent treatment /of verapamil & beta-blockers/ in those with impaired left ventricular function could be dangerous if...a 10-15% depression in myocardial function takes place. /Salt not specified/
...Absolute contraindications to the use of verapamil (the acute stage of myocardial infarction, complete atrioventricular block, cardiogenic shock, overt heart failure)...should not be injected together with a beta-adrenergic blocking agent, or within 3 times the half-life of that agent. /Salt not specified/
The basic physiologic actions of verapamil may lead to serious adverse effects. /Salt not specified/
Maternal Medication usually Compatible with Breast-Feeding: Verapamil: Reported Sign or Symptom in Infant or Effect on Lactation: None. /from Table 6/ /Salt not specified/
For more Drug Warnings (Complete) data for Verapamil (23 total), please visit the HSDB record page.

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Pharmacodynamics
Verapamil is an L-type calcium channel blocker with antiarrhythmic, antianginal, and antihypertensive activity. Immediate-release verapamil has a relatively short duration of action, requiring dosing 3 to 4 times daily, but extended-release formulations are available that allow for once-daily dosing. As verapamil is a negative inotropic medication (i.e. it decreases the strength of myocardial contraction), it should not be used in patients with severe left ventricular dysfunction or hypertrophic cardiomyopathy as the decrease in contractility caused by verapamil may increase the risk of exacerbating these pre-existing conditions.

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2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile is a tertiary amino compound that is 3,4-dimethoxyphenylethylamine in which the hydrogens attached to the nitrogen are replaced by a methyl group and a 4-cyano-4-(3,4-dimethoxyphenyl)-5-methylhexyl group. It is a tertiary amino compound, an aromatic ether, a polyether and a nitrile.
Verapamil is a phenylalkylamine calcium channel blocker used in the treatment of high blood pressure, heart arrhythmias, and angina, and was the first calcium channel antagonist to be introduced into therapy in the early 1960s. It is a member of the non-dihydropyridine class of calcium channel blockers, which includes drugs like [diltiazem] and [flunarizine], but is chemically unrelated to other cardioactive medications. Verapamil is administered as a racemic mixture containing equal amounts of the S- and R-enantiomer, each of which is pharmacologically distinct - the S-enantiomer carries approximately 20-fold greater potency than the R-enantiomer, but is metabolized at a higher rate.
Verapamil is a Calcium Channel Blocker. The mechanism of action of verapamil is as a Calcium Channel Antagonist, and Cytochrome P450 3A4 Inhibitor, and Cytochrome P450 3A Inhibitor, and P-Glycoprotein Inhibitor.
Verapamil is a first generation calcium channel blocker used for treatment of hypertension, angina pectoris and superventricular tachyarrhythmias. Verapamil has been linked to a low rate of serum enzyme elevations during therapy and to rare instances of clinically apparent acute liver injury.

Verapamil has been reported in Teichospora striata and Schisandra chinensis with data available. LOTUS - the natural products occurrence database Verapamil is a phenylalkylamine calcium channel blocking agent. Verapamil inhibits the transmembrane influx of extracellular calcium ions into myocardial and vascular smooth muscle cells, causing dilatation of the main coronary and systemic arteries and decreasing myocardial contractility. This agent also inhibits the drug efflux pump P-glycoprotein which is overexpressed in some multi-drug resistant tumors and may improve the efficacy of some antineoplastic agents.
VERAPAMIL is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 1981 and has 4 approved and 16 investigational indications.

Verapamil is only found in individuals that have used or taken this drug. Verapamil is a calcium channel blocker that is a class IV anti-arrhythmia agent. [PubChem]Verapamil inhibits voltage-dependent calcium channels. Specifically, its effect on L-type calcium channels in the heart causes a reduction in ionotropy and chronotropy, thuis reducing heart rate and blood pressure. Verapamil's mechanism of effect in cluster headache is thought to be linked to its calcium-channel blocker effect, but which channel subtypes are involved is presently not known. [PubChem] Calcium channel antagonists can be quite toxic. In the management of poisoning, early recognition is critical. Calcium channel antagonists are frequently prescribed, and the potential for serious morbidity and mortality with over dosage is significant. Ingestion of these agents should be suspected in any patient who presents in an overdose situation with unexplained hypotension and conduction abnormalities. The potential for toxicity should be noted in patients with underlying hepatic or renal dysfunction who are receiving therapeutic doses. (A7844).
A calcium channel blocker that is a class IV anti-arrhythmia agent.

C27H38N2O4.HCL

491.06

490.259

C, 66.04; H, 8.01; Cl, 7.22; N, 5.70; O, 13.03

152-11-4

Verapamil;52-53-9;Verapamil-d3 hydrochloride;Verapamil-d6 hydrochloride;1185032-80-7;Verapamil-d3-1 hydrochloride;2714485-49-9

62969

White to off-white solid powder

1.058g/cm3

586.1ºC at 760 mmHg

142 °C (dec.)(lit.)

308.3ºC

5.895

1

6

13

34

606

0

Cl[H].O(C([H])([H])[H])C1=C(C([H])=C([H])C(=C1[H])C(C#N)(C([H])([H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C([H])([H])C1C([H])=C([H])C(=C(C=1[H])OC([H])([H])[H])OC([H])([H])[H])C([H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])[H]

DOQPXTMNIUCOSY-UHFFFAOYSA-N

InChI=1S/C27H38N2O4.ClH/c1-20(2)27(19-28,22-10-12-24(31-5)26(18-22)33-7)14-8-15-29(3)16-13-21-9-11-23(30-4)25(17-21)32-6;/h9-12,17-18,20H,8,13-16H2,1-7H3;1H

2-(3,4-dimethoxyphenyl)-5-[2-(3,4-dimethoxyphenyl)ethyl-methylamino]-2-propan-2-ylpentanenitrile hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 5 mg/mL (10.18 mM) (saturation unknown) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.24 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.24 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (4.24 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 25 mg/mL (50.91 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)